Immunoassays are widely used as methods for qualitatively or quantitatively measuring the presence of an analyte existing in a biological sample such as urine or blood. Of these immunoassays, an immunochromatography method is generally used with high frequency since its implementation is simple and enables short-time measurement.
The competitive reaction and the sandwich reaction are broadly used as immunoreactions to be employed in immunochromatography methods. In particular, the sandwich reaction is mainly employed for an immunochromatography method. In a typical example of the use of the sandwich reaction, the following procedures are performed to detect an analyte comprising an antigen in a sample. (1) A chromatographic medium having a reaction zone is prepared by immobilizing a fine particle as a solid phase fine particle that has been sensitized with an antibody against an antigen that is an analyte on a chromatographic medium or by directly immobilizing the antibody on a chromatographic medium. (2) Meanwhile, a sensitization-target fine particle is prepared by sensitizing a labeled fine particle with an antibody capable of specifically binding to an analyte. (3) The sensitized and labeled fine particle is caused to migrate chromatographically on a chromatographic medium together with a sample.
The thus immobilized antibody is as an immobilized reagent at the reaction zone formed on the chromatographic medium by the above procedures. The sensitized and labeled fine particle specifically binds to the reagent via an antigen that is an analyte. As a result, the presence, absence, or the amount of an analyte in a sample is measured by visually determining the presence, absence, or the degree of signals generated when the sensitized and labeled fine particle is captured at the reaction zone.
In such immunochromatography method, colloidal metal particles or colloidal metal oxide particles, colloidal nonmetal particles, and dye particles are used as fine particles for preparation of labeled fine particles. Moreover, an enzyme such as alkaline phosphatase or peroxidase may be used as a label substance.
In the case of some immunochromatography methods, detection signals are amplified to avoid the problem of no antigens being detected because of low sensitivity (false negative). As such a signal amplification method, there is a method using an enzyme such as alkaline phosphatase or peroxidase as a label substance. There is also a method of detecting an analyte by sensitization using a silver-containing compound and a reducing agent used for silver ions as label substances selected from the group consisting of metal colloid label substances and metal sulfide label substances.
When the signal of a label substance is amplified, there is a case where the signal of another label substance that exists outside the detection line of an immunochromatographic strip is also amplified and background noise is thereby increased. This problem can be solved by allowing an amplification solution to directly come into contact with the detection line or by allowing the amplification solution to come into contact with a portion extremely close to the detection line.
The immunochromatography methods are described in JP Patent Publication (Kokai) No. 2002-202307 A, JP Patent Publication (Kohyo) No. 10-513263 A (1998) and U.S. Pat. No. 7,189,522.
Among bioactive substances or environmental pollutants such as natural products, toxins, hormones, or agricultural chemicals, numerous substances act in ultratrace amounts. Accordingly, instrumental analytical methods capable of performing high-sensitivity analysis have conventionally been widely used for qualitative and quantitative measurement of these substances. However, instrumental analytical methods are poor in specificity, require excessive time for analysis including pretreatment of samples, and are troublesome in operation. Thus instrumental analytical methods are inconvenient for the purpose of rapid and convenient measurements that have been required in recent years. Meanwhile, immunoassays are highly specific and much easier in terms of operation than instrumental analytical methods. Therefore immunoassays have gradually spread in the field of measurement of bioactive substances and environmental pollutants. However, conventional immunoassays such as enzyme immunoassays and latex agglutination assays using 96-well plates do not always provide satisfactory rapidness and convenience for measurement or detection sensitivity.
Another need expected to be enabled is as follows. Achievement of higher sensitivity of tests that currently use relatively invasive samples such as swabs and blood makes it possible to detect very small amounts of analytes contained in relatively low-invasive samples such as snot, mouth wash, and urine. Thus, less burdensome tests of patients can be realized.
In recent years, test kits using an immunochromatography method (hereinafter referred to as an immunochromatography kit) have been used more often in examination of infections that require particularly rapid diagnosis. According to the spread of these kits, patients with infections can be identified by a rapid and convenient method, and subsequent diagnosis and therapy can be conducted immediately and accurately. For example, in an immunochromatography method using the sandwich method, a labeled first antibody capable of specifically binding to an analyte (for example, an antigen) and a sample solution which may possibly contain the analyte are developed on a first insoluble carrier (for example, a nitrocellulose membrane, a glass fiber membrane, a nylon membrane, cellulose membrane, etc.) on which a second antibody capable of specifically binding to the analyte has been immobilized in a specific region. As a result, an immune complex with the analyte is formed in the region on which the second antibody of the first insoluble carrier has been immobilized. The analyte can be measured by detecting a signal such as color development or coloring of a label substance. The label substance to be used herein may be, for example, a protein such as an enzyme, colored latex particles, metal colloids, or carbon particles.
The immunochromatography method requires neither massive facilities nor instruments for determination and measurement. Furthermore, the immunochromatography method is simple in operation and promptly gives measurement results by introducing a sample solution dropwise which may possibly contain an analyte and leaving it for approximately 5 to 10 minutes. For this reason, this technique is used widely as a convenient, rapid, and highly specific method for determination and measurement in many scenarios, such as for clinical examination in hospitals and in assays in laboratories.
Among bioactive substances or environmental pollutants such as natural products, toxins, hormones, and agricultural chemicals, many substances exert effects in ultratrace amounts that are undetectable by conventional common immunochromatography methods. Therefore, there are demands for development of rapid, convenient, and highly sensitive immunochromatography methods for such substances.
As immunochromatography methods for achieving high sensitivity by signal amplification, an amplification method using enzymes (Japanese Patent No. 3237540, and Japanese Patent No. 3309977), a method involving chemical amplification (Japanese Patent No. 3886000), and a method involving silver amplification (JP Patent Publication (Kokai) No. 2002-202307 A) have been known. When a signal is amplified, noise caused by a label substance existing in the background is also simultaneously amplified. Thus, in order to eventually increase detection sensitivity, it is particularly necessary to decrease noise caused by such a label substance existing in the background. In Japanese Patent No. 3309977, a washing solution is supplied to wash such a label substance in the background in an enzyme amplification immunography method. However, this washing operation is not sufficient for carrying out an examination with sensitivity higher than that of a common enzyme amplification immunochromatography method, towards which the present system is directed.
As a method for increasing the sensitivity of an immunochromatography method using a metal colloid as a label substance, a silver amplification method has been known (JP Patent Publication (Kokai) No. 2002-202307 A). In JP Patent Publication (Kokai) No. 2002-202307 A, after a solution has been supplied, another solution used for silver amplification is added dropwise to a detection line portion to carry out amplification, thereby achieving high sensitivity. However, if high sensitivity that is above a certain level is required, an increase in noise caused by a label substance existing in the background becomes problematic.
Like the present system, when signal amplification is carried out using an amplification solution used for silver amplification that enables amplification at a level higher than that of the solution used in silver amplification disclosed in JP Patent Publication (Kokai) No. 2002-202307 A, if detection sensitivity is intended to be increased, further noise reduction is necessary.
Under the present circumstances, in any case, a technique of sufficiently decreasing noise, which is required when signal amplification is carried out using an amplification solution used for silver amplification that enables high-level amplification, has not yet been introduced.